Upon immune activation, naive CD4+ T cells differentiate into one of several functionally specialized types of helper T cells that coordinate immune responses through the selective expression of cytokine genes. Proper regulation of helper T cell lineage decisions is critical for effective immunity against pathogens and for the prevention of inappropriate responses that underlie allergic and autoimmune diseases. Epigenetic regulation and well-characterized transcription factor networks control the extensive reprogramming of gene expression that occurs during T cell differentiation. More recently, it has become clear that microRNAs (miRNAs) also play a critical role in this process. T cells lacking all miRNAs exhibit reduced cell proliferation and survival, as well as abnormally rapid and unrestrained differentiation. Thus, one of the limiting roles of miRNAs in naive T cells is to restrain the gene expression changes that lead to differentiation. This is particularly intriguing in light of our preliminary data that indicate that T cell activation induces potent posttranscriptional downregulation of all miRNAs compared with other cellular RNAs, including bulk mRNAs and ribosomal RNAs. Several highly expressed miRNAs appear to be further downregulated by reduced transcription of the primary transcripts from which they are processed. We hypothesize that activation signals trigger T cell differentiation in part by inducing widespread miRNA downregulation. The long-term goals of the proposed research are to discover how these changes in miRNA expression are controlled, and to reveal how they affect helper T cell differentiation and gene expression. The specific aims are: i) To define the mechanism(s) that modulate miRNA homeostasis in activated T cells by profiling miRNA processing intermediates and biogenesis pathway protein expression, and by defining the signaling requirements for activation-induced miRNA downregulation;ii) To dissect the transcriptional and epigenetic regulation of miRNA genes in T cells through examination of chromatin remodeling and transcription factor binding, and iii) To elucidate the functional consequences of miRNA regulation by modulating the expression of individual miRNAs and the activity of transcriptional and posttranscriptional regulators of miRNA homeostasis in activated T cells. Identifying these mechanisms and their functional consequences will lead to a better understanding of the molecular control of T cell responses, and may lead to novel strategies for immunomodulation or immunosuppression. PUBLIC HEALTH RELEVANCE: Helper T cells are the central coordinators of immune responses. Recent discoveries in the field of gene regulation uncovered an entire class of small regulatory RNA molecules, called microRNAs that had previously escaped detection. MicroRNAs play a critical role in shaping the immune function of helper T cells, and the expression of the microRNAs themselves is dramatically altered during T cell responses. The goal of this project is to uncover the molecular mechanisms that underlie these processes. These studies may lead to novel approaches for vaccine development, or for designing therapies for asthma and other allergic diseases, autoimmune diseases, and chronic inflammation.